Andrew H. Wyllie

Defying death after DNA damage.

DNA damage frequently triggers death by apoptosis. The irreversible decision to die can be facilitated or forestalled through integration of a wide variety of stimuli from within and around the cell. Here we address some fundamental questions that arise from this model. Why should DNA damage initiate apoptosis in the first place? In damaged cells, what are the alternatives to death and why should they be selected in some circumstances but not others? What signals register DNA damage and how do they impinge on the effector pathways of apoptosis? Is there a suborganellar apoptosome complex effecting the integration of death signals within the nucleus, just as there is in the cytoplasm? And what are the consequences of failure to initiate apoptosis in response to DNA damage?

Where, O death, is thy sting? A brief review of apoptosis biology.

Apoptosis was a term introduced in 1972 to distinguish a mode of cell death with characteristic morphology and apparently regulated, endogenously driven mechanisms. The effector processes responsible for apoptosis are now mostly well known, involving activation of caspases and Bcl2 family members in response to a wide variety of physiological and injury-induced signals. The factors that lead of the decision to activate apoptosis as opposed to adaptive responses to such signals (e.g. autophagy, cycle arrest, protein synthesis shutoff) are less well understood, but the intranuclear Promyelocytic Leukaemia Body (PML body) may create a local microenvironment in which the audit of DNA damage may occur, informed by the extent of the damage, the adequacy of its repair and other aspects of cell status.

Heterogeneity studies identify a subset of sporadic colorectal cancers without evidence for chromosomal or microsatellite instability

Two apparently independent mechanisms of instability are recognized in colorectal cancer, microsatellite instability and chromosomal instability. Evidence from colorectal cancer cell lines indicates the presence of either, or both, types of instability in the vast majority. Here, we sought to determine the prevalence of such instability in primary sporadic colorectal cancers. Microsatellite instability was established by demonstration of ovel clonal, nongerm-line alleles in at least two of four tested loci. Chromosomal abnormalities were identified by comparative genomic hybridization (CGH) and flow cytometric analysis of nuclear DNA content. Tumours harbouring chromosomal instability were distinguished from those with stable but aneuploid karyotypes by comparing chromosomal defects at multiple sites throughout each cancer. This analysis allowed assessment of both the number of chromosomal abnormalities and their heterogeneity throughout the tumour. The results confirm that microsatellite instability is consistently associated with multiple, repeated changes in microsatellites throughout the growth of the affected colorectal carcinomas. There were also several carcinomas in which major structural or numerical abnormalities in chromosomes had clearly continued to arise during tumour growth. However, a substantial subset of tumours showed neither microsatellite instability nor multiple, major chromosomal abnormalities. We suggest that the development of a proportion of colorectal cancers proceeds via a different pathway of carcinogenesis not associated with either of the currently recognized forms of genomic instability.

Apoptosis: the germs of death

From the initial recognition that programmed cell suicide existed, to the elucidation of the underlying death and survival pathways at the molecular level, the story of apoptosis has unfolded rapidly. But much still remains to be discovered.

New insights into the role of PML in tumour suppression

The PML gene is involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), which generates the oncogenic fusion protein PML (promyelocytic leukaemia protein)-retinoic acid receptor alpha. The PML protein localises to a subnuclear structure called the PML nuclear domain (PML-ND), of which PML is the essential structural component. In APL, PML-NDs are disrupted, thus implicating these structures in the pathogenesis of this leukaemia. Unexpectedly, recent studies indicate that PML and the PML-ND play a tumour suppressive role in several different types of human neoplasms in addition to APL. Because of PMLs extreme versatility and involvement in multiple cellular pathways, understanding the mechanisms underlying its function, and therefore role in tumour suppression, has been a challenging task. In this review, we attempt to critically appraise the more recent advances in this field and propose new avenues of investigation.

Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression

Background and aims Although aberrant methylation of key genes in the progression of colorectal neoplasia has been reported, no model-based analysis of the incremental changes through the intermediate adenoma stage has been described. In addition, the biological drivers for these methylation changes have yet to be defined. Linear mixed-effects modelling was used in this study to understand the onset and patterns of the methylation changes of SFRP2, IGF2 DMR0, H19, LINE-1 and a CpG island methylator phenotype (CIMP) marker panel, and they were correlated with DNA methyltransferase 3B (DNMT3B) levels of expression in a sample set representative of colorectal neoplastic progression. Methods Methylation of the above CpG islands was measured using quantitative pyrosequencing assays in 261 tissue samples. This included a prospective collection of 44 colectomy specimens with concurrent normal mucosa, adenoma and invasive cancer tissues. Tissue microarrays from a subset of 64 cases were used for immunohistochemical analysis of DNMT3B expression. Results It is shown that the onset and pattern of methylation changes during colorectal neoplastic progression are locus dependent. The CIMP marker RUNX3 was the earliest CpG island showing significant change, followed by the CIMP markers NEUROG1 and CACNA1G at the hyperplastic polyp stage. SFRP2 and IGF2 DMR0 showed significant methylation changes at the adenomatous polyp stage, followed by the CIMP markers CDKN2A and hMLH1 at the adenocarcinoma stage. DNMT3B levels of immunohistochemical expression increased significantly (p<0.001) from normal to hyperplastic and from adenomatous polyps to carcinoma samples. DNMT3B expression correlated positively with SFRP2 methylation (r=0.42, p<0.001, 95% CI 0.25 to 0.56), but correlated negatively with IGF2 DMR0 methylation (r=0.26, p=0.01, 95% CI −0.45 to −0.05). A subset of the CIMP panel (NEUROG1, CACNA1G and CDKN2A) positively correlated with DNMT3B levels of expression (p<0.05). Conclusion Hierarchical epigenetic alterations occur at transition points during colorectal neoplastic progression. These cumulative changes are closely correlated with a gain of DNMT3B expression, suggesting a causal relationship.

Early-onset colorectal cancer with stable microsatellite DNA and near-diploid chromosomes

Colorectal cancer has been described in terms of genetic instability selectively affecting either microsatellite sequences (MIN) or chromosome number and structure (CIN). A subgroup with apparently stable, near-diploid chromosomes and stable microsatellites (MACS) also exists. These distinctions are important, partly because of their value in highlighting different pathways of carcinogenesis, and partly because of their direct relevance to prognosis. Study of early-onset cancer has often proved a fruitful resource for the identification of the nature and function of cancer susceptibility genes. In a study of colorectal cancer with stable microsatellite DNA, we describe 22 early-onset tumours (mean age=33), compared with 16 late-onset tumours (mean age=68). Both groups contained carcinomas with the MACS phenotype, characterized by near diploid DNA content, as defined by flow cytometry, and minimal chromosome arm deletion or amplification (six or less events per genome), determined by comparative genomic hybridization (CGH). Minimal chromosome imbalance correlated strongly with diploid DNA content (P<0.001). The proportion of MACS cancers was significantly greater in early-onset as compared to late-onset tumours (64 vs 13%, P=0.005). Of the chromosome arm imbalances commonly observed in late-onset tumours, only 18q− was observed more than twice amongst the 14 early-onset MACS tumours. Seventy-nine per cent of these MACS tumours were located in the distal colon, and 69% were at advanced clinico-pathological stages (with lymph node or distant metastasis). A positive family history of colorectal or other cancers was elicited in seven patients in the MACS early-onset group, and one additional patient in this group had a metachronous ovarian cancer. The results suggest that MACS cancer may have a genetic basis different from either MIN or CIN, and further studies of these cancers may lead to discovery of new mechanisms of colorectal carcinogenesis and cancer susceptibility.

Prognostic relevance of DNA copy number changes in colorectal cancer.

In a study of 109 colorectal cancers, DNA copy number aberrations were identified by comparative genomic hybridization using a DNA microarray covering the entire genome at an average interval of less than 1 Mbase. Four patterns were revealed by unsupervised clustering analysis, one of them associated with significantly better prognosis than the others. This group contained tumours with short, dispersed, and relatively few regions of copy number gain or loss. The good prognosis of this group was not attributable to the presence of tumours showing microsatellite instability (MSI‐H). Supervised methods were employed to determine those genomic regions where copy number alterations correlate significantly with multiple indices of aggressive growth (lymphatic spread, recurrence, and early death). Multivariate analysis identified DNA copy number loss at 18q12.2, harbouring a single gene, BRUNOL4 that encodes the Bruno‐like 4 splicing factor, as an independent prognostic indicator. The data show that the different patterns of DNA copy number alterations in primary tumours reveal prognostic information and can aid identification of novel prognosis‐associated genes. Copyright

Caspase-mediated cleavage of APC results in an amino-terminal fragment with an intact armadillo repeat domain

During the effector phase of apoptosis, caspase activation appears to be responsible for the distinctive structural changes of apoptosis and perhaps for some of the changes in function of the doomed cells. There is therefore interest in identifying caspase substrates and the details of the cleavage events. Here we define precisely the event responsible for generation of a stable 90 kDa fragment from the oncosuppressor protein adenomatous polyposis coli (APC). Using synthetic radiolabeled APC peptides as substrate, we demonstrate cleavage by cytosolic extracts from preapoptotic cells. This cleavage was reproduced by recombinant caspase‐3 and blocked by a tetrapeptide inhibitor Ac‐DEVD‐CHO, which is specific for caspase‐3 family members. Inhibitors specific for caspase‐1 and ‐8 however, were less effective in blocking APC cleavage. Mutation of a candidate DNID caspase‐3 target site completely abolished cleavage. This cleavage may be of biological importance since the 90 kDa fragment consists of a sequence that is highly conserved in the human, rat, mouse, Xenopus, and Drosophila APC, although wide sequence divergence is observed in Drosophila immediately carboxy‐terminal to the DNID site. Furthermore, cleavage at this site separates two significant functional domains: an aminoterminal armadillo repeat and an adjacent series of β‐catenin binding sites. Further circumstantial evidence for the significance of APC‐related pathways in apoptosis is provided by the observation that apoptosis also induces cleavage of β‐catenin itself, a protein known to accumulate in cells depleted in functional APC and that appears to link cell–cell signaling to changes in transcription and cell movement.—Webb, S. J., Nicholson, D., Bubb, V. J., Wyllie, A. H. Caspase‐mediated cleavage of APC results in an amino‐terminal fragment with an intact armadillo repeat domain. FASEB J. 13, 339–346 (1999)

Evidence for the receipt of DNA damage stimuli by PML nuclear domains.

Promyelocytic leukaemia nuclear domains (PML‐NDs) comprise a shell of PML protein and many labile cargo proteins. The nature of their cargo, their juxtaposition to foci of damaged DNA following ionizing radiation (IR), and the altered DNA damage responses in PML null cells all implicate PML‐NDs in the DNA damage response. In this work, the propensity of PML‐NDs to increase in number and decrease in size following IR has been studied. Serial quantitative studies of endogenous PML‐NDs prove that the PML‐ND response to IR is not the result of the asymmetry in cell cycle distribution that can follow IR, but reflects more directly the process of DNA damage. The response is swift, sensitive (evident after 1 Gy), and potentially reversible in untransformed fibroblasts. In these cells and in HCT116 colon cancer cells, failure to restore PML‐ND number within 24 h correlates with later loss of growth potential—in fibroblasts, through prolonged cell cycle arrest and in HCT116 cells, through apoptosis. Failure to express an intact ATM/CHK2 DNA damage signalling pathway in either cell type leads to a delay in the PML‐ND response to IR. Conversely, cell cycle progression following IR in cells that detect damaged DNA accelerates PML‐ND reorganization. Collectively, these data show that the increase in PML‐ND number seen after irradiation is, in part, triggered by the receipt of the DNA damage stimulus. The senescent cell state is also associated with chronic DNA damage and Hayflick‐limited fibroblasts were found to express nuclei with elevated numbers of PML‐NDs before IR that remained unresponsive to IR. Though the underlying reasons for damage‐induced PML alteration remain obscure, it is noteworthy that significant numbers of PML‐NDs juxtapose with ionizing radiation‐induced foci after IR. The co‐regulation of these structures may necessitate the stereotyped increases in PML‐ND number following damage. Copyright